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OPEN Efect of earthworms on mycorrhization, root morphology and biomass of silver fr seedlings inoculated with black summer trufe ( aestivum Vittad.) Tina Unuk Nahberger 1, Gian Maria Niccolò Benucci 2, Hojka Kraigher 1 & Tine Grebenc 1*

Species of the Tuber have gained a of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in trufe production is to infect , , beech, etc. in greenhouse conditions. We aimed to show whether silver fr (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with trufes, and how earthworms in the inoculation substrate would afect the mycorrhization dynamics. Silver fr seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms afect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting efects on fne root biomass and morphology of silver fr seedlings. Only a few of the assessed fne root parameters showed statistically signifcant response, namely higher fne root biomass and fne root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fne root tip density when earthworms were added, the specifc root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative efect of earthworm on branching density. Silver fr was confrmed as a suitable host partner for commercial mycorrhization with trufes, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The efect of earthworms on mycorrhization of silver fr with was positive only after 6 months of mycorrhization, while this efect disappeared and turned insignifcantly negative after 12 months due to the secondary efect of grazing on ectomycorrhizal root tips.

Hypogeous fungi are a diverse polyphyletic group of over 150 globally distributed genera 1 that form sequestrate fruiting bodies 2,3. Among hypogeous fungi, trufes, the genus Tuber P.Micheli ex F.H.Wigg., family 4, are recognized as a gastronomic delicacy due to their unique -specifc aromas 5–7. Due to this charac- teristic, trufes are a signifcant commercial product among non-timber forest products 8. Trufes are ectomycorrhizal 9, ectendomycorrhizal 10 or endophytic fungi 11 that are unable to complete their life cycle without a symbiotic relationship with a vital plant host 3,12. Trufes grow in an ectomycorrhizal symbio- sis with most boreal and temperate trees, and with perennial shrubs of the northern hemisphere, such as , hazel, beech, birch, pines, pecan, cistus, sunrose, etc. 13–17. Te only known example of forming ectendomycor- rhizae is with the strawberry tree 10. High cultural, gastronomic, and economic interest in trufes guided many attempts of trufe cultivation. However, cultivation of commercially valuable species has gained higher interest

1Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, Slovenia. 2Department of Plant, Soil, and Microbial Sciences, Michigan State University, 426 Auditorium Road, East Lansing, MI 48824, USA. *email: tine.grebenc@ gozdis.si

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only in the last few decades 13,14,18. Today we can successfully cultivate most commercially valuable trufe species with a range of host plants using trufe-inoculated seedlings 19,20. A routine spore-inoculation process of seedlings in greenhouses has remained mainly unchanged for decades 21. A pasteurized substrate with added pre-treated trufe spores is used for inoculation of seedlings. Seedlings are then maintained in nurseries until ectomycor- rhizae are established 21–23. Current trufe mycorrhization procedures do not suggest additional treatments such as microbiota or pedofauna enrichments of substrate, nor was this ever tested under nursery conditions (ibid.). Te trufe life cycle is limited to soil 24. While mycelia actively grow in soil, move mainly together with the roots, and soil surface conidia are wind dispersed 25, sexual spores move by soil fauna, such as large and small mammals, insects, earthworms, etc. for dispersal 26. Gange 27 found that earthworms graze preferentially on soils containing mycorrhizal fungal propagules and fruiting bodies, and disperse propagules and spores by soil ingestion or as particles attached to their cuticles 28. Earthworms profoundly impact the between mycorrhizal fungi and plants not only directly by grazing and moving fungal propagules in soil but also indirectly via changing soil permeability and modifying nutrient availability 28–31. In nature earthworms preferentially feed on fungal mycelia, consequently afecting the mycelium either by disrupting the contact of the external hyphae from the roots, decreasing fungal biomass in soil or reducing hyphal length in substrate 28,29. Tuber aestivum Vittad. (the summer trufe) is a trufe species with a very broad ecological niche 32 and consequently one of the most geographically widespread trufes in Europe 2,18,33. Tuber aestivum is also one of the less demanding species that forms ectomycorrhizae with a broad range of ectomycorrhizal partners 13,34 thus making it easy and successfully cultivated widely, in Europe as far north as Sweden and Finland 14,21. Although Pico (the white Italian trufe) is regarded as the most famous and expensive trufe, in Italy alone over 65% of the traded and processed trufes are Tuber aestivum 35. Silver fr (Abies alba Mill.) is an ectomycorrhizal evergreen conifer of mountainous regions of eastern, western, southern, and central Europe 36. Silver fr is a long-lived tree with of great ecological and silvicultural value (ibid.) known to form ectomycorrhizae with a range of fungal genera. Past studies revealed the presence of the genus Tuber in mycorrhizae with silver fr, but infrequently and in low percentages 37–40. Silver fr in this study was selected as a plant partner tree due its ability to form ectomycorrhizae with trufes in nature, which has never been confrmed in a nursery inoculation process, and never with a commercial trufe species. Based on the roles of earthworms in soil, and contradictory statements about their efects on mycorrhization, we performed a greenhouse inoculation experiment to analyze their efects on mycorrhization and fne root growth. For this purpose, we inoculated silver fr seedlings with Tuber aestivum to test if silver fr could be an adequate ectomycorrhizal host for commercial trufes. We set the following hypotheses: (1) spore inoculation, earthworms or a combination of both will have a signifcant efect on silver fr root biomass and morphology; (2) silver fr can be an adequate symbiotic partner for commercial production of Tuber aestivum mycorrhized seedlings; and (3) earthworms added to the mycorrhization system will have a positive efect on mycorrhization levels of silver fr with the commercial trufe species Tuber aestivum under greenhouse conditions. Materials and methods Pot experimental design. Silver fr seedlings were obtained from the LIECO nursery (LIECO GmbH & Co KG, Kalwang, Austria) and were approximately four months old at the beginning of the experiment. Seed- lings of about the same size and with well-developed above and belowground parts were used for the experi- ment. A check of randomly selected ten silver fr seedlings confrmed the absence of any contaminant ectomyc- orrhizal fungi prior to inoculation. Te substrate for mycorrhization used in the experiment was adapted from 21,41. Te fnal volumes of pas- teurized components in substrate mixture was: black peat (50 vol.%) (Agrocentre Roko d.o.o., Hoče, Slovenia), vermiculite (33 vol.%) (Njiva d.o.o., Ložnica pri Savinje, Slovenia), perlite (17 vol.%) (Knauf Gips KG, Iphofen, Germany). and ground limestone (2 vol.%) (Rotar d.o.o., Dobrova, Slovenia). Prior to adding Tuber aestivum spore suspension, substrate was well watered, and pH was adjusted with additional lime dust to a range from 7.0 to 7.5. Inoculation substrate was supplemented with a trufe spore suspension containing 2 g fresh weight of mature trufe gleba per seedling­ 2. Fresh trufes were washed, surface sterilized in 70% ethanol for 10 min and washed under running tap water for 20 min. Finally, peridium was peeled and gleba was kept frozen (− 20 °C) until preparation of the suspension. Only high quality, fully ripened (> 95% asci with ripened spores), mechanically undamaged, and unrotten sporocarps of Tuber aestivum were used. Quality and identifcation of each sporocarp was confrmed by a trufe specialist, and reference sporocarps are kept in the LJF herbarium under accession numbers TUBAES/290915 (under hornbeam and oak; Žlebič, Slovenia), TUBAES/231114A (under beech and silver fr; Snežna jama, Slovenia) and TUBAES/050714A (under beech and silver fr; Mavrovo, North Mac- edonia). To obtain the desired concentration of 2 g Tuber aestivum spores per plant, 200 g of sporocarps were homogenized with a surface sterilized blender in sterilized water. Te spore suspension was added to substrate in adequate volume and the inoculation substrate was mixed thoroughly by hand. 25 silver fr seedlings were planted in individual containers with dimensions 52 cm × 38 cm × 50 cm. Tese containers were used instead of traditional pots (340 ml/650 ml) pots to ensure sufcient living space for added earthworms since traditional pots were proved insufcient for their survival. For the container experiment we established four treatments: control treatment, treatment with added trufe inoculum, treatment with added earthworms, and treatment with a combination of trufe inoculum and earthworms. One earthworm per seedling was added approximately one month afer the experiment was initiated. Eisenia fetida (Savigny, 1826) earthworms were obtained from a local farm in Slovenia, where animals of approximately the same size (ca 4 cm long) were selected for the experi- ment. Containers with diferent treatments were kept in a fully controlled greenhouse for one year. Controlled

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conditions with a day light regime and temperature 22 °C for 16 h and a night regime and temperature of 15 °C for 8 h were maintained throughout the experiment.

Evaluation of mycorrhization levels and assessment of fne root morphology. Fine root myc- orrhization was evaluated 6 and 12 months afer inoculation, which is the standard time for mycorrhization success control 19. Ten seedlings were randomly selected from each treatment per evaluation time and carefully removed from containers. Whole root systems of selected plants were washed in tap water, fne roots were sepa- rated and placed in trays flled with distilled water and scanned by an Epson Perfection V700 Photo scanner (Seiko Epson Corp., Suwa, Nagano, Japan). Fine roots were defned as roots with diameter < 2 mm 42. Scans were further analyzed using WinRHIZO sofware (Regent Instruments Inc., Québec City, Canada) following the protocol in 43. Te total root length, number of root tips, and number of branches per individual seedling were assessed and the following rations were calculated: (1) fne root length: fne root biomass (SRL—specifc root length), (2) number of root tips: root biomass (root tip density), (3) number of root tips: fne root length (specifc root tip density), and (4) number of branches: fne root biomass (branching density). Fine root systems biomass was assessed (SCALTEC SBC-31 balance; Denver Instrument, Bohemia, NY, USA) afer drying at 70 °C. Ectomycorrhizae from each of 100 seedlings were identifed following morphological criteria in 44–46 under a binocular (Olympus SZH, Tokyo, Japan) and a microscope (Zeiss AxioImager Z2, Carl Zeiss Microscopy GmbH, Jena, Germany) following the procedure described in Fischer and Colinas 47. First the complete root system was cut longitudinally, and one half was chosen for examination. Roots from the chosen half were further cut in 2–3 cm segments and placed over a 1 cm × 1 cm grid in a large petri dish flled with distilled water. In con- secutively random chosen grid squares, all fne roots were separated into non-mycorrhizal root tips (N), Tuber aestivum ectomycorrhizal root tips (T) and contaminant ectomycorrhizal root tips (O). Ectomycorrhizal tips in each category were counted until the total sum per seedling reached 250 ectomycorrhizal tips. Old and senescent ectomycorrhizal tips were categorized as non-mycorrhizal root tips. Based on 250 counted ectomycorrhizal tips the proportion of Tuber aestivum ectomycorrhizae (PT = T/(total number of ectomycorrhizal tips)), and the proportion of contaminant ectomycorrhizae (PO = O/(total number of ectomycorrhizal tips)) were calculated. Seedlings from each treatment and for each sampling time were subjected to a standardized quality control certifcation procedure to evaluate the suitability of the batch for commercialization. Te quality control followed 48, according to which a certifed plant must show at least 25% of all fne roots mycorrhized with the desired truf- fe species, and the level of contaminant ectomycorrhizal fungi must not exceed 25% of all fne roots. Seedlings from each treatment and for each sampling time were also evaluated following criteria proposed by Fischer and Colinas 47, where the minimum colonization by the desired trufe species should be at least 10% of all fne roots, with a level of contaminant ectomycorrhizal fungi not higher than 50%.

Molecular analysis. Most trufe mycorrhized certifcation procedures also require a DNA-based confrma- tion of the identity 19. To fulfll this criterion, ectomycorrhizal tips from diferent morphotypes were subjected to molecular identifcation. DNA extractions were performed with a DNeasy Plant Mini kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Te nuclear rDNA ITS region was amplifed from isolated DNA using the specifc primer pair ITS1F and ITS4 49,50 and ITS5 and ITS7 51. Te PCR reactions with the primer pair ITS1f/ITS4 were performed as described in Grebenc and Kraigher 52, while the PCR reactions with the primer pair ITS5/ITS7 were carried out under the following conditions: a denaturation step of 7 min at 95 °C, followed by 32 cycles of 15 s at 94 °C, 15 s at 56 °C and 40 s at 72 °C, with a fnal step at 72 °C for 7 min. PCR products were run on 1.5% agarose gels in 0.5 × TBE bufer and visualized with Gel Doc EQ System, PC (BioRad, ZDA). Amplifed DNA fragments were cut out of agarose gels and purifed with the innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany) following manufacturer’s instructions. Afer the DNA fragments purifcation, sequencing was performed at the commercial sequencing laboratory Mac- rogen (Macrogen Europe B.V., Amsterdam, Te Netherlands). All samples were sequenced in both directions with the primers ITS1f/ITS4 49,50 or ITS5/ITS7 51. Te obtained sequences were processed in Geneious version 11.1.4 (https​://www.genei​ous.com, Kearse et al. 53. Te BLASTN algorithm from the NCBI website (National Center for Biotechnology Information; https​://blast​.ncbi.nlm.nih.gov/Blast​.cgi) was used to assess the similarity of obtained ITS sequences to sequences in GenBank. Representative sequences from ectomycorrhizal root tips were submitted to GenBank.

Statistical analysis. All statistical analyses were performed with R 3.5.1 54. Normal distribution and homo- geneity of variance were tested with the Shapiro–Wilk normality test and the Bartlett test, and were improved with the approach of Tukey’s Ladder of Powers where needed (p > 0.05), from the package “rcompanion”. For statistical evaluation of diferences between treatments, two-way analysis of variance (ANOVA) was applied to test the efects of time from spore inoculation and treatment description as independent factors and their inter- action. Further one-way ANOVA was used to analyze the efects of added inoculum, earthworms, and the com- bination of both on root biomass and root morphology, separately per time from trufe spore inoculation. As a post-hoc test, the Tukey HSD test was used. Te efect of earthworms on mycorrhization with Tuber aestivum was tested with Student’s t-test using a two-sample unequal variance (heteroscedastic), afer the normal distribu- tion was confrmed and data variance of both treatments was confrmed as signifcantly diferent afer an F test.

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Results Efects of mycorrhization, earthworms or combination of both treatments on silver fr fne root biomass and morphology. Te diferent inoculation treatments revealed contrasting efects on fne root biomass and morphology of silver fr seedlings. Only a few of the assessed parameters showed statistically signifcant diferent efects when compared to the performance of the control plants (Supplement Table 1). Mycorrhization with Tuber aestivum, addition of Eisenia fetida earthworms to mycorrhization substrate, and the combination of both treatments resulted in signifcant diferences in fne root morphology and biomass (Fig. 1). Fine root biomass was signifcantly higher only in Tuber aestivum mycorrhized plants 6 months afer inoculation (Fig. 1A). Tis efect became insignifcant afer 12 months, and only in seedlings mycorrhized with Tuber aestivum and with the addition of Eisenia fetida a pronounced increase was noted (Fig. 1B). Te Tukey HSD test showed no signifcant diferences in specifc fne root length neither 6 nor 12 months afer inoculation (Fig. 1C,D, respectively). Te fne root tip density was signifcantly lower in earthworm- and trufe-inoculated seedlings 6 months afer inoculation (Fig. 1E). Afer 12 months in control, the fne root tip density decreased, and highest number of fne root tips was observed in Tuber aestivum mycorrhized plants (Fig. 1F). Specifc root tip density 6 months afer inoculation was highest in control plants and insignifcantly dropped in all other treat- ments (Fig. 1G). 12 months afer inoculation the specifc root tip density remained the same in control plants while it increased signifcantly in inoculated seedlings and insignifcantly in treatments involving earthworms (Fig. 1H). Te ratio between number of branches and fne root biomass expressed as branching density (number of root forks/mg of fne roots) was higher in both treatments where silver fr seedlings were inoculated with Tuber aestivum. 6 months (Fig. 1I) and 12 months (Fig. 1J) afer inoculation, the highest number was calculated for treatments with Tuber aestivum mycorrhized seedlings followed by combined treatment of Tuber aestivum and earthworms. A negative efect of earthworm grazing on branching density was also observed between Tuber aestivum mycorrhized seedlings and seedlings that were inoculated solely with earthworms.

Silver fr as a host for the commercial trufe species Tuber aestivum. Mycorrhization levels and quality control of silver fr plants mycorrhized with Tuber aestivum were checked 6 and 12 months afer spore inoculation. Te mycorrhization level distribution falls between 6% and 35% (mean = 6.47% ± 5.1%) 6 months from inoculation and between 36% and 55% (mean = 37.72% ± 14.04%) 12 months from inocula- tion. Te level of contaminant ectomycorrhizae 6 months afer spore inoculation was low, between 7% and 13% (mean = 8.01% ± 5.05%) in Tuber aestivum inoculated seedlings. 12 months afer spore inoculation the level of contaminant fungi in Tuber aestivum inoculated seedlings was even lower, between 0% and 3% (mean = 0.65% ± 1.2%) for 50% of all trufe inoculated seedling samples (Fig. 2). Mycorrhization levels of seedlings inoculated with Tuber aestivum evaluated by 48 criteria for a trufe inocu- lated seedlings certifcation revealed that 6 months afer inoculation 20% of Tuber aestivum inoculated silver fr seedlings passed the criteria of a minimum 25% of mycorrhized root tips. 12 months afer spore inoculation, the share of Tuber aestivum inoculated silver fr seedlings passing the minimum required criterion was 80%. Considering the criteria proposed by Fischer and ­Colinas47 all silver fr seedlings inoculated with Tuber aestivum met criteria 12 months afer spore inoculation (Fig. 2). All inoculated seedlings showed well-developed Tuber aestivum ectomycorrhizae (Fig. 3A,B) with typical pseudoparenchymatous outer mantle layers in the ectomycor- rhizal mantle (Fig. 3C). Sequences from morphotypes showed 100% identity with Tuber aestivum/uncinatum, and contaminating ECM belonging to the genus Telephora are available in GenBank under accession numbers MN270339–MN270351 and MN270390–MN270412.

Efect of earthworms on mycorrhization with Tuber aestivum. Te addition of earthworms to Tuber aestivum inoculated silver fr seedlings (Fig. 4) resulted in a signifcantly higher mycorrhization percent- age in seedlings 6 months afer inoculation (Student’s t-test p = 0.025; F test for homogeneous variances p = 0.125, kurtosis = 1.809 (no earthworms)/− 1.458 (with earthworms) and skewness = 1.542 (no earthworms)/0.268 (with earthworms)). 12 months afer inoculation the efect of added earthworms turned into insignifcantly negative (Student’s t-test p = 0.096; F test for homogeneous variances p = 0.117, kurtosis = − 1.115 (no earthworms)/− 1.319 (with earthworms) and skewness = 0.072 (no earthworms)/− 0.108 (with earthworms)). Te reduction of the mycorrhization level in 12-month-old seedlings was at least partially due to grazing of ectomycorrhizal fne roots by earthworms (Fig. 5) that was not observed earlier in the experiment. All negative control plants showed zero mycorrhization level with Tuber aestivum. Discussion Tuber aestivum spore inoculation efect on fne root biomass and morphology. Efects of ecto- mycorrhizal fungi on fne roots morphology and branching 55–60 are well established, but very few studies have included efects of earthworms on fne roots and mycorrhization. Our results recoded 6 and 12 months afer sil- ver fr inoculation with ectomycorrhizal fungi and earthworms did not give straightforward results. Te fne root biomass increase was signifcant only for mycorrhized seedlings without earthworms in frst 6 months. Similar phenomenon was already observed by while the insignifcant positive efect of earthworms could be explained by their active roles in nutrient mixing and nutrient-releasing efects 61. As another early efect, the fne root tip density was higher in control plants 6 months afer inoculation and signifcantly lower in earthworm-inoculated plants. Tis early peak is explained by the relatively little time that passed from inoculation to analysis by ecto- mycorrhizal fungi and/or earthworms. Tese results indicate that the Tuber aestivum mycorrhization process in silver fr is among slower ones, similar to mycorrhization of non-typical Tuber aestivum ectomycorrhizal host plants, such as pecan 6,23, and in contrast to more pioneer species like hazel that are mycorrhized faster 62,63. Te slower mycorrhization of silver fr with Tuber aestivum was also confrmed by the failure to fulfll the certifca-

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Figure 1. Efects of mycorrhization with Tuber aestivum, Eisenia fetida earthworms and a combination of both treatments on silver fr root biomass and fne root morphology parameters afer 6 and 12 months of mycorrhization in containers under controlled conditions; n = 10. Te signifcantly diferent values afer the Tukey HSD post-hoc test (p < 0.05) are annotated with a diferent lowercase letter.

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Figure 2. Mycorrhization levels (%) of silver fr plants inoculated with Tuber aestivum and control plants, 6 and 12 months afer the start of the experiment. Te fgure represents a boxplot based on % of mycorrhized fne roots based on ten seedlings per treatment. Outliers are shown with circles.

Figure 3. Tuber aestivum root tips under dissecting microscope (A, B) and root tip outer-middle mantle under microscope (C).

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Figure 4. Mycorrhization level of Tuber aestivum inoculated seedling with and without added earthworms, afer 6 months and 12 months from spore inoculation, n = 20.

Figure 5. Consequences of Tuber aestivum mycorrhized ectomycorrhizal fne roots grazed by earthworms. Ectomycorrhizal fne roots which were partially or completely grazed were regarded as old and senescent ectomycorrhizal root tips and were counted as non-mycorrhizal root tips.

tion criteria 6 months afer inoculation of seedlings. On the other hand, 12 months afer inoculation, the lowest number for fne root tip density was expectedly observed in control plants where no branched ectomycorrhizal roots were formed, in contrast to Tuber aestivum mycorrhized plants with abundant monopodial-pinnate to monopodial-pyramidal branching (Fig. 3A,B). Another parameter with a signifcant change was the specifc fne root tip density 12 months afer inoculation. Specifc fne root tip density was higher in mycorrhized seedlings only, and not in treatments with added earthworms, which is again related to the changed morphology of ecto- mycorrhizal fne roots compared to a non-mycorrhized fne roots 64. All other morphological parameters, regardless of the duration of experiment, showed no signifcant deviation from control plants and among parameters. Te low responsiveness of morphological root parameters to mycor- rhization with Tuber aestivum and earthworms is due to silver fr being a slow-growing, shade-tolerant long-living conifer tree species of predominantly mountainous regions 36, opposite to Tuber aestivum as a fast-growing pioneer species of ruderal areas with dynamic soil conditions­ 18,32,65. We have also recorded an insignifcant efect of earthworms on fne root biomass of non-mycorrhized seedlings. Tis “lack of interest” of earthworms in bare fne roots is likely due to the presence of repelling resins in fne roots as in other parts of silver fr 66,67.

Silver fr is an appropriate host partner for T. aestivum cultivation. We have successfully estab- lished ectomycorrhizae of Tuber aestivum on silver fr in greenhouse conditions, using a standard commercial inoculation procedure 21,23,41. Ectomycorrhizae of Tuber aestivum with silver fr had not been confrmed before and we managed to produce seedlings that passed two common procedures for certifcation of trufe-inoculated seedlings 47,48 at least afer 12 months of mycorrhization. Te successful mycorrhization with Tuber aestivum was

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expected, since trufes are known to form ectomycorrhizae with a wide range of mycorrhizal plant partners such as Betula, Carpinus, Castanea, Cedrus, Corylus, Fagus, Ostrya, Picea, Pinus, Pecan, Quercus, Tilia 2,3,13,46,68. Te mycorrhization level of silver fr was comparable to angiosperm hosts used commercial mycorrhization 19. Based on our results, satisfactory mycorrhization rates, i.e. at least 25% all fne roots mycorrhized with the desired trufe species 48, were only achieved afer 12 months of mycorrhization. Tis suggests that commercial mycor- rhization should be adapted to this time frame, or better mycorrhization conditions ought to be sought. Quality mycorrhized seedlings are not sufcient for cultivation of trufes with desired plant partners unless climate, soil, and cultivation conditions for both partners in symbiosis are met at the level to favor their growth and fruiting 16. Fortunately, silver fr 36 and Tuber aestivum 18,32 are tolerant to a wide range of soil conditions, nutrient content and availability as well as pH levels, making their cultivation plausible. A confrmation that suitable conditions can be met in nature is the example of beech-silver fr dominated forests covering the Dinaric Alps, where Tuber aestivum was collected in suitable micro locations 69–73. High altitude limestone mountain chains are also poten- tial areas for future Tuber aestivum cultivation with silver fr or in combination with other host partners.

Efect of earthworms on mycorrhization of silver fr with the commercial trufe species Tuber aestivum. Te efects of earthworms on mycorrhization of silver fr with Tuber aestivum were ambiguous. Te early efect afer 6 months was positive, either due to perturbation or improved spore germination. Te efect turned insignifcantly negative afer 12 months. Inconsistency in efects were previously reported by sev- eral authors who recorded either positive, neutral or negative efects on mycorrhizal root colonization 29,30,74. Due to the low number of studies, we can only speculate on possible mechanisms behind the efects. Te early positive efect on mycorrhization could be attributed to stimulation of fungal growth due to enhanced nutrient and organic matter mineralization and substrate perturbation caused by earthworms 28,30. Mixing of substrate where spores patchy rather than evenly distributed, could favor mycorrhization. Te late (e.g. 12 months afer inoculation) negative efect on mycorrhization was predominantly due to grazing on already mycorrhized fne roots. Grazing of mycorrhized fne roots, but not non-mycorrhized roots, is due to a combination of reasons, for example, nutrient limitation in mycorrhization substrate 28, low appreciation of resin-rich silver fr bare roots, and the fact that mycelium of fungi is nutrient rich and as such a tempting substrate for hungry earthworms 65. Based on our results we cannot give a clear management suggestion for application of earthworms in truf- fe mycorrhization procedures. Te early efect on mycorrhization and general efects on substrate mixing and nutrients availability is positive, while longer mycorrhization times under the highly nutrient-limited environ- ment of mycorrhization pots may cause it to become negative, as was already observed by 75–78 Bringing together experiences from negative and positive efects to modify the mycorrhization process may be a solution that is worth further investigation. Conclusions In this study we revealed a signifcant efect of mycorrhization on root biomass and morphogenesis, as signif- cantly higher seedling root tip density and specifc root tip density compared to control seedlings was observed afer 1 year from spore inoculation, fndings which confrm Hypothesis 1. Hypothesis 2 can also be confrmed, as the observed results revealed that silver fr is an appropriate plant host for commercial cultivation of Tuber aestivum trufes, as inoculated silver fr seedlings passed the criteria or parameters which are defned for cer- tifcation of seedlings mycorrhized with Tuber species. Lastly, we hypothesized that earthworms would have a positive impact on mycorrhizal root colonization as was reported by some authors; however, in our study earth- worms had a positive short-term efect while the efect turned negative due to the secondary efect of grazing on ectomycorrhizal root tips, thus Hypothesis 3 remains unresolved. Data availability Reference collections for trufe samples used in the mycorrhization process are deposited at the LJF ofcial herbarium. Original observations dataset generated and analyzed during the current study are available from the corresponding author on reasonable request.

Received: 16 August 2020; Accepted: 2 March 2021

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Mycorrhizal colonization and nitrogen uptake by maize: Combined efect of tropical earthworms and velvetbean mulch. Biol. Fertil. Soils 44, 181–186 (2007). 78. Reddell, P. & Spain, A. V. Earthworms as vectors of viable propagules of mycorrhizal fungi. Soil Biol. Biochem. 23, 767–774 (1991). Acknowledgements Te authors acknowledge lab assistance from Barbara Štupar and Melita Hrenko (Slovenian Forestry Institute). Trufes for inoculation were collected by Žarko Volk and Dr. Tine Grebenc (Slovenia), and by Toni Mitrov and Prof. Dr. Mitko Karadelev (North Macedonia). Anonymous reviewers are acknowledged for their contribution to improvement of the manuscript in the revision process. Author contributions Te work is a part of the PhD thesis of T.U.N., under the supervision of T.G. Te experiment was set up by T.U.N., T.G., G.M.N.B., and H.K. Lab analyses and statistics were done by T.U.N. Te manuscript was drafed by T.U.N. All authors read and approved the fnal manuscript. Funding Tis research was supported by the Young Researcher Scheme (for Tina Unuk Nahberger), research project J4-1766 “Methodology approaches in -based diversity and ecological plasticity study of trufes from their natural distribution areas” and the Research Program in Forest Biology, Ecology and Technology (P4- 0107), all of the Slovenian Research Agency. Te study was also supported by the LIFEGENMON project (LIFE ENV/SI/000148).

Competing interests Te authors declare no competing interests.

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